Noroviruses, previously called Norwalk-Like Viruses (NLV), are small round viruses within the calicivirus family and are important viral pathogens that cause acute gastroenteritis, the second most common illness in the United States. Norwalk disease is a mild to moderate illness that develops 1-2 days after infection by person-to-person transmission, surface contamination, or by contaminated food or water and the illness lasts for 24-60 hours. Symptoms include nausea, vomiting, diarrhea, abdominal pain and upon occasion headache and low fever. Severe illness requiring hospitalization is most unusual. Particularly large epidemic outbreaks of illness have occurred following consumption of contaminated water or uncooked food such as salad and ham, and shellfish including clams, cockles, and oysters.
Since the molecular cloning of noroviruses and the subsequent development of new diagnostic assays, noroviruses have been recognized as the most important cause of non-bacterial epidemics of acute gastroenteritis in both developed and developing countries, affecting individuals of all ages. Over 90% of non-bacterial gastroenteritis outbreaks in the United States are found to be associated with noroviruses. The percentage of individuals in developing countries who have antibodies against noroviruses at an early age is very high (as it is with poliovirus and other viruses transmitted by fecal contamination of water and foods). In the United States the percentage increases gradually with age, reaching 50% in people over 18 years of age. The antibody prevalence to noroviruses is estimated to be higher based on the data using new diagnostic methods than previously reported using old methods. Immunity, however, is not permanent, and re-infection can occur.
Although the Norwalk Virus (NV), the prototype norovirus strain, was discovered in 1972, knowledge about the virus has remained limited because it has failed to grow in cell cultures and no suitable animal models have been found for virus cultivation. Human stool samples obtained from outbreaks and from human volunteer studies, therefore, are the only source of the virus. Still the concentration of the virus in stool is typically so low that virus detection with routine electron microscopy is not possible. Although limited studies on human volunteers have shown that antibody response to norovirus challenge is protective against subsequent infection, other studies have found that pre-existing antibodies against norovirus are not protective. Even more puzzling has been that some individuals with high levels of antibody against norovirus were more susceptible to the virus than individuals who did not have the antibody, and that 20-30% of individuals who did not have antibody against norovirus could not be infected by challenge with norovirus.
Before molecular cloning of NV in 1990, several methods were developed for diagnosis that included immune electron microscopy and other immunologic methods such as radio immunoassays (RIAs) or enzyme linked immunoabsorbent assays (ELISAs). Because most of these methods use reagents from infected humans that are of limited source, they are not widely used. Since the molecular cloning of NV, several methods have been developed for diagnosis, including reverse transcription-polymerase chain reaction (RT-PCR) for detection of the viral RNA and recombinant enzyme immune assay for detection of viral antigens and antibody against viral antigens. Due to the wide genetic and antigenic diversity of noroviruses, these methods encountered problems of low detection sensitivity and specificity.
Because a tissue culture model for human norovirus infection is not available, recombinant molecular approaches have been necessary to dissect their basic biological properties. The genome of NV has been cloned, sequenced, and characterized biochemically. The genome is a single-stranded, positive-sense RNA of about 7.5 kb and is organized into three open reading frames (ORF's). ORF1 encodes the non-structural proteins such as polymerase, protease and helicase. ORF2 encodes a major capsid protein, and ORF3 encodes a minor structural protein.
Noroviruses are morphologically similar but genetically and antigenically diverse viruses. Genetically, noroviruses can be divided into two main genogroups, the “Norwalk-like viruses” (NLVs) and the “Sapporo-like viruses” of human caliciviruses, a subset of Caliciviridae. Each genogroup can be further divided into genetic clusters or genotypes. The major human strains can be genetically divided into at least 15 genetic clusters. The ORF2-encoded major capsid protein self-assembles into virus-like particles (VLPs) when the recombinant capsid protein is expressed in high concentrations from recombinant baculoviruses in insect cells. Norovirus VLPs are morphologically and immunologically indistinguishable from the wild-type viruses found in human feces, and have provided the foundation for studies of assembly, attachment, and immunobiology. Moreover, they have been indispensable for the development of diagnostic immunoassays.
Despite the lack of a tissue culture system or an animal model that accurately depicts human norovirus disease, significant advances in norovirus attachment and pathogenesis research have been achieved, including the identification of particular ABH (ABO) histo-blood group antigens as putative ligands for the attachment of noroviruses to mucosal cell surfaces. These carbohydrate epitopes are present in mucosal secretions and on many tissues throughout the human body. The complexity of the ABH histo-blood group antigens is reflected in the large number of type chain branches and modifying enzymes, as well as the broad distribution of different antigens based on blood type, genetic polymorphisms, and tissue specificity.
Although the ABH antigens are typically described as blood group antigens because of their presence on red blood cells, they are also found on other tissues, and may be more appropriately termed histo-blood group antigens (HBGAs). In the blood they exist in both a cellular form on platelets and a soluble form as blood group active glycosphingolipids coupled to plasma lipoproteins. They exist as membrane antigens on such diverse cells as vascular endothelial cells and intestinal, cervical, urothelial, pulmonary and mammary epithelial cells. Soluble forms are also found in various secretions and excretions, such as saliva, milk, urine, and feces. In some tissues, their appearance is developmentally regulated. Despite their wide distribution, genetic inheritance, developmental regulation, and importance in transfusion and transplantation, their normal physiological function, if any, remains a mystery.
At least five type chains of the ABH histo-blood group antigens have been identified, which are produced and distributed in various tissue compartments in a type-specific manner. The type 1 and 3 chain molecules are the predominant ABH histo-blood group antigens on cell surfaces of the gut, which is presumed to be the site of norovirus infection. The FUT2 enzyme, an α1,2-fucosyltransferase, is responsible for the production of type 1 and 3 chain ABH histo-blood group antigens. Approximately 20% of Europeans and Africans express a defective form of the FUT2 enzyme and therefore do not produce type 1 or 3 chain histo-blood group antigens on cell surfaces of the gut or in mucosal secretions; this is known as the “nonsecretor” or secretor-negative phenotype.
The type 1 and 3H antigens are produced by the FUT2 enzyme in secretor-positive individuals from H precursor molecules. The A or B enzymes produced in blood type A or B individuals, respectively (or both in blood type AB individuals), further modify the H antigens along the particular type chain to produce type-specific A or B antigens. Similarly, the type 2 chain A or B antigens are produced on red blood cells, and the presence or absence of one or more of these molecules is used to determine an individual's blood type. In the type 1 chain, the FUT3 Lewis enzyme, an α1,4-fucosyltransferase, modifies the H type 1 precursor to produce Lea or modifies H type 1 to produce Leb. Approximately 10% of Europeans lack a functional FUT3 gene product, representative of the Lewis-negative phenotype, and therefore will not produce Lea or Leb on mucosal surfaces. Secretor-positive, Lewis-positive individuals will produce Leb and relatively less Lea on cell surfaces of the gut and in mucosal secretions, whereas secretor-negative, Lewis-positive individuals will produce only Lea.
Recombinant NV VLPs were first shown to attach to H types 1 and 3 epitopes on the surfaces of gastroduodenal epithelial cells from secretor-positive individuals. In addition, Norwalk VLPs attach specifically to H type 1, H type 3, and Leb carbohydrates, even in the absence of other cellular components. The significance of these in vitro findings was recently demonstrated in vivo using a human challenge model for Norwalk virus infection. This study demonstrated that secretor-negative human volunteers, who do not produce H type 1, H type 3, and/or Leb on mucosal cell surfaces, were resistant to live NV challenge. Taken together, the results of these studies suggest that H type 1, H type 3, and Leb are attachment molecules necessary for NV infectivity in the gut.
It has also been observed that VLPs of other norovirus strains exhibit different properties of attachment to ABH histo-blood group antigens in vitro, suggesting that different norovirus genotypes may utilize different mechanisms for attachment and entry in vivo and that various other host factors may play a role in susceptibility to different norovirus strains. Because VLP reagents are not available for many norovirus strains, the overall extent to which noroviruses utilize ABH histo-blood group antigens for attachment is unclear and cannot be thoroughly evaluated using previously described biochemical methods. Also, no study to date has examined and compared the ability of wild-type virus and recombinant VLPs to attach to specific ABH histo-blood group antigens.
In the present invention, a method was developed to purify noroviruses from clinical specimens and at the same time further characterize their histo-blood group antigen attachment properties. Consistent with previous observations, various norovirus strains exhibited distinct histo-blood group antigen binding patterns. Ultimately the study of noroviruses presents the need, among other things, to understand the specific mechanism for norovirus infection within the GI tract, the specific binding properties of the prototype NV with the ABO blood antigens and the Le blood antigens, the specific binding properties of the other noroviruses with the human histo-blood phenotypes and their respective blood antigens, and the compounds and compositions that are effective to inhibit binding between noroviruses and blood antigens. The ultimate goal of current norovirus research is to prevent or treat an infection by a norovirus and/or the resulting illness.
Virus like particles (VLPs) of the various noroviruses can recognize and bind to one or more human histo-blood group antigens, and human histo-blood group antigens can recognize and bind to one or more noroviruses, in varied HBGA-norovirus binding patterns. Thus, the present invention is premised on the notion that noroviruses can infect humans who have a particular human histo-blood type that presents blood antigens that can bind the particular strain of infecting norovirus, and further that a strain of norovirus will bind with one or more HBGAs, but will not bind with all other blood group antigens. It would therefore be advantageous to understand the specific binding properties of the various norovirus strains with the ABO blood antigens and the Lewis blood antigens by characterizing the norovirus strain-specificity to human HBGAs. It would also be advantageous to provide compounds and compositions that are effective to bind specific, small regions of the norovirus capsid protein, such that a kit can be developed to determine if an individual is susceptible to infection by a particular norovirus strain.